Glycosylation of proteins is the most common and diverse form of post-translational modification. It can profoundly affect the function of glycoproteins in normal and pathological states, for example in cell-cell adhesion, fertilization, inflammation and malignant transformation (Varki et al., 2009). Most human tumor cells express glycoproteins with aberrant glycosylation patterns. The most studied cancer-specific glycans are truncated O-glycans such as the T-nouvelle antigen (Tn; GalNAcα) and the Thomsen-Friedenreich pancarcinoma tumor-associated carbohydrate antigen (TFα, Galβ1-3GalNAcaα1-Ser/Thr), which uniquely decorate mucin-type glycoproteins in about 90% of human cancer cells, but are distinctly absent from normal tissues except for the placenta in early pregnancy. The presence of such cancer-specific glycans can indicate increased invasiveness and metastatic potential.
Carbohydrate-binding proteins have therefore enormous utility as tools to monitor the expression of glycans for a wide range of basic research and clinical applications. For instance, a number of carbohydrate-binding lectins and mammalian antibodies have been used to detect expression of tumor-associated carbohydrate antigens for diagnostic and prognostic purposes (Powlesland et al., 2009; Li et al., 2010; Almogren et al., 2012). Glycan-binding proteins can also be used for a variety of in vivo applications, such as targeting specific cells and tissues for imaging, for drug delivery, and to control carbohydrate-mediated processes. Unfortunately, most readily available glycan-binding proteins, such as plant and animal lectins, and mammalian antibodies, typically display either broad reactivity, poor specificity, or both. For example, most monoclonal antibodies (mAb) that specifically recognize the Thomsen-Friedenreich antigen are of the IgM isotype, which are large antibodies with relatively low affinity, and therefore have limited clinical utility (Almogren et al., 2012). Furthermore, carbohydrate-binding proteins are only available for a tiny fraction of over 7,000 glycan determinants estimated for the human glycome (Cummings, 2009). Therefore, methods to generate tailored glycan-binding proteins with high affinity and selectivity for any glycan of interest could revolutionize the field.
A number of strategies have been evaluated for obtaining glycan-binding receptors. The most commonly used approach involves immunizing animals with an appropriate glycan or glycoconjugate to raise mAbs (Rittenhouse-Diakun et al., 1998; Li et al., 2010), but this process can be slow and labor intensive. This approach works well in certain cases, but many interesting glycans are conserved among species and, therefore, are non-immunogenic. Other approaches include directed evolution of lectins (Powlesland et al., 2009; Hu et al., 2012) and of single chain Fv antibodies (scFv) (Ravn et al., 2007; Sakai et al., 2010), small peptide carbohydrate receptors (Boltz et al., 2009) and carbohydrate-binding aptamers (Sun et al., 2010), but these have not proven to be general methods and have not gained widespread use. Therefore, a simple, efficient, and general method is still critically needed.